Methods of predicting mortality risk by determining telomere length

ABSTRACT

The present invention provides for methods of determining telomere length of an organism and correlating the measured telomere length with mortality risk associated with telomere length in a population. The presence of shorter telomeres is associated with an increased mortality rate and increased susceptibility to certain types of diseases for an individual member of a human population.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/442,456, filed Jan. 24, 2003, the entire contents of which isincorporated herein by reference.

TECHNICAL FIELD

The present invention relates to methods of predicting mortality rates,and in particular to methods for determining the length of telomeres andcorrelating telomere length with survival, mortality risk, andsuceptibility to age related diseases, such as cardiovascular disease,neoplastic disease, and infectious disease. The invention furtherrelates to use of the methods to identify individuals or groups ofindividuals at risk of developing such age related diseases.

BACKGROUND OF THE INVENTION

Telomeres are specialized structures present at the ends of lineareukaryotic chromosomes that function in replicating and maintaining theintegrity of the chromosomal ends. Telomeres distinguish the chromosomalterminus from other types of double stranded breaks responsible forinitiating cell growth arrest and aberrant chromosomal fusions.

Telomeric sequences vary between species, but their essential featuresare similar between eukaryotes. The telomeric DNA is generally composedof tandem repeats of a basic sequence unit. Telomeric repeats of someorganisms are perfect repeats, such as sequence TTAGGG seen in humans orslime mold. Others, such as those of yeast or protozoans, have irregularrepeat sequences. In general, the G rich strand runs 5′ to 3′ to thechromosomal terminus. The length of the repeat sequences range from afew to tens of kilo base pairs.

In some organisms, the characteristic telomeric repeat sequences areabsent and are substituted by other sequences that function similar totelomere repeat sequences. For example, in Drosophila melanogaster, thetelomeres are generally composed of non-LTR-type retrotransposons,called HeT-A and TART elements. In the mosquito Anopheles gambiae, thechromosomal ends are composed of arrays of complex sequence tandemrepeats rather than short repeat sequences.

In some organisms, a 3′ single stranded overhang is present at theterminus of the telomere. The length of the single stranded region isvariable, extending from about 100 bp or more. Under defined conditionsin vivo or in vitro, the overhanging terminus associates with varioustelomere associated proteins and invades the double stranded telomereregion to form t-loop structures (Griffith, J. D. et al., Cell 97:503-514 (1999); Munoz-Jordan et al., EMBO J. 20: 579-588 (2001)).Formation of t-loop structures is believed to function in negativelyregulating telomere elongation by telomerase, and in addition, provide amechanism for sequestering the single stranded ends to protect them fromdegradation and to suppress activation of DNA damage checkpointpathways.

Typically, the DNA replicative machinery acts in the 5′ to 3′ direction,and synthesis of the lagging strand occurs discontinuously by use ofshort RNA primers that are degraded following strand synthesis. Sincesequences at the 3′ end of a linear DNA are not available to completesynthesis of the region previously occupied by the RNA primer, theterminal 3′ region of the linear chromosome is not replicated. This “endreplication problem” is solved by the action of telomerase, a telomerespecific ribonucleoprotein reverse transcriptase. The telomerase enzymehas an integral RNA component that acts as a template for extending the3′ end of the telomere. Repeated extensions by telomerase actitivtyresults in the generation of telomere repeats copied from thetelomerase-bound RNA template. Following elongation by telomerase,lagging strand synthesis by DNA polymerase completes formation of thedouble stranded telomeric structure.

In normal human somatic cells, telomerase is not expressed or expressedat low levels. Consequently, telomeres shorten by 50-200 bp with eachcell division until the cells reach replicative senescence, at whichpoint the cells looose the capacity to proliferate. This limitedcapacity of cells to replicate is generally referred to as the Hayflicklimit, and may provide cells with a counting mechanism, i.e., a mitoticclock, to count cell divisions and regulate cellular development.Correspondingly, activation of telomerase in cells lacking telomeraseactivity, for example by expressing telomerase from a constituteretroviral promoter or activation of endogenous polymerase, allows thecells to maintain proliferative capacity and leads to immortalization ofthe cell.

Interestingly, these immortalized cells have short stable telomereswhile the shortest telomeres become extended. This phenomena suggeststhat telomerase enzyme protects short telomeres from further shorteningwhile extending those that have fallen below a certain threshold length.Thus, presence of telomerase activity does not appear to be necessarywhen telomeres are a certain length, but becomes critical to maintenanceof telomere integrity when it falls below a critical limit.

It is well established that the length and integrity of telomeres isimportant for proper segregation of chromosomes and cell growth. Forexample, development of many types of cancers correlates with activationof telomere maintenance while cell senescence correlates with loss oftelomere integrity. Shortening of telomere induced by inhibitingtelomerase activity can lead to proliferative senescence and cellapoptosis (Zhang, X. et al., Genes Dev. 2388-99 (1999)). Moreover,genetic knockouts of telomerase RNA in mice results in animals withdevelopmental defects, age related pathologies, and increased cancersusceptibility (Rudolph, K. L. et al., Cell 96: 701-12 (1999); Herrera,E. et al., EMBO J. 18: 2950-60 (1999)). Similarly, in the autosomaldominant disorder of dyskeratosis congenita (DKC), which arises from amutation in the gene encoding the RNA component of telomerase, afflictedpatients display accelerated telomere shortening and die at a median ageof 16 years (maximum approximately 50 years), usually from severeinfections secondary to bone marrow failure. Clinical features of DKCpatients, further suggestive of accelerated aging, include prematuregraying and loss of hair; skin dyspigmentation; poor wound healing; highrisk of severe infections; and an increased incidence of malignancies,osteoporosis, and pulmonary fibrosis. In addition, the shortest averagetelomere lengths measured in blood DNA from normal elderly individualsoverlap with the highest average telomere lengths measured in blood fromDKC patients.

In view of the role telomeres play in cell growth and cell senescence,it is desirable to have methods of predicting the occurrence of agerelated diseases and mortality risk based on length of telomeres. Thiswill provide a basis for identifying individuals with increased risk ofdeveloping particular age-associated diseases, such as cancer andhypertension, such that early medical intervention can be administeredto individuals in high risk groups. In addition, these methods can beused to identify genetic and environmental factors that may play a rolein changing the aging process or altering disease susceptibility inindividuals and in populations.

SUMMARY OF THE INVENTION

In accordance with the above objects of the invention, the presentinvention provides methods of determining telomere length andassociating the measured telomere length with a mortality risk orlikelihood of disease occurrence that corresponds to a telomere lengthobserved in a population.

Various methods for measuring telomere length are provided, includingmeasuring mean terminal restriction fragment (TRF), quantitativefluorescent in situ hybridization, flow cytometry, and polymerase chainreaction. In a preferred embodiment, the telomere length is measuredusing PCR and telomere specific primers designed to amplify telomererepeat sequences. This provides a rapid method to measure averagetelomere length within a cell or population of cells and is particularlysuited for large-scale population studies.

In one embodiment, the telomere length is measured from somatic cells,particularly those that show low or no levels telomerase activity.Suitable samples are derived from blood, and particularly the lymphoidcells in the blood.

The telomere length is determined for an individual and correlated withtelomere length observed in a population. The mortality rate within thepopulation is assessed relative to telomere length. In a preferredembodiment, the population is aged matched with the age of theindividual organism being examined. For humans, the age-matchedpopulation is within about 10 years of the age of the individual, morepreferable within 5 years, and most preferable within one year.

Correlation of the measured telomere length of the individual and thepopulation is examined by various statistical methods, such as survivalanalysis, including Cox proportional hazard regression models,Kaplan-Meier survival distribution estimate, Peto Wilcoxon test, maximumlikelihood analysis, multiple regression analysis, and others.

In another embodiment, the mortality risk is determined for rate oftelomere shortening and mortality within a population. The rate at whichthe telomere shortens for an individual is measured to determine themortality risk to that individual.

In another embodiment, the methods of the present invention is used topredict the likelihood of occurrence of age related diseases in apopulation and individuals within the population. Age related diseasesthat may be examined, include, but are not limited to, cardiovasculardisease (e.g., hypertension, myocardial infarction, stroke, etc.),neoplastic diseases (e.g., cancers, carcinomas, malignant tumors, etc.),and susceptibility to infectious diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the sequence of the oligonucleotide primer pairs, tel-1(SEQ ID NO:1) and tel-2 (SEQ ID NO:2), used to amplify human telomericrepetitive units. Shown are the hybridization schemes of the primers tothe telomere repetitive sequences (SEQ ID NO: 3 and 4) and hybridizationof the primers to each other. The tel-1 primer can hybridize to anyavailable complementary 31 basepair stretch along the strand oftelomeric DNA oriented 5′ to 3′ toward the centromere. The tel-2 primercan hybridize to any complementary 33 basepair stretch along the strandoriented 5′ to 3′ toward the end of the chromosome. For each primer,nucleotide residues are altered to produce mismatches between thealtered residue and nucleotide residues at the identical nucleotideposition of each repetitive unit to which the primer hybridizes. Thus,for tel-1 and tel-2, every sixth base is mismatched. To limitprimer-dimer products, the altered residues of each primer also producesa mismatch with the 3′ terminal nucleotide residue of the other primerwhen the primers hybridize to each other, thus blocking extension bypolymerase. In addition, the 5′ terminal regions of the primer aredesigned so as to not basepair with the telomeric repeats. Thesenoncomplementary 5′ terminal sequences prevent the 3′ ends of the PCRproducts from initiating DNA synthesis in the middle of telomereamplification products.

FIG. 2 shows standard curves used to measure relative T/S ratios, wherethe T/S ratio is the telomere (T) to single copy gene (S) ratio (seeExample 2). Five DNA concentrations over an eight-fold range weregenerated by serial dilution (dilution factor ˜1.68) and aliquoted intomicrotiter plate wells; the final amounts per well ranged from 12.64 ngto 100 ng, with the middle quantity approximately matching that of thesamples being assayed. The C_(t) of a DNA sample is the fractionalnumber of PCR cycles to which the sample must be subjected in order toaccumulate enough products to cross a set threshold of magnitude offluorescent signal. Any individual or pooled human DNA sample may beused to create the standard curves, as long as each assayed sample'sC_(t) falls within the range of C_(t)'s of the standard curves. O=singlecopy gene 36B4; Δ=telomere.

FIG. 3 shows the correlation of relative T/S ratios determined by realtime quantitative PCR using the primers described herein and the meantelomere restriction fragment (TRF) lengths determined by traditionalSouthern hybridization analysis. The DNA samples used for the analysiswere from blood drawn from 21 individuals. All relative T/S ratiosplotted have values ≦1.0 because the initial T/S ratios determined usingthe standard curves have all been normalized to the lowest T/S ratio(0.69) observed among the samples. The equation for the linearregression line best fitting the data is shown.

FIG. 4 shows association of telomere length (TL) in blood DNA after age60 with subsequent survival. The graph shows the proportion of theoriginal sample of research subjects who remained alive (y axis) atvarious time points (x axis) after the blood draw. In each panel“longer” identifies individuals from the top half of the TLdistribution, and “shorter” identifies individuals from the bottom halfof the distribution: a, both sexes combined; b, women.; c, men. Data areplotted as fitted survival curves, according to the group prognosismethod (Ghali, W. A. et al., JAMA 286: 1494-1497 (2001), incorporatedherein by reference).

FIG. 5 shows association of telomere length (TL) in subjects aged 60-74years, and in subjects aged 75 years or older, with subsequent survival.The graph shows the proportion of the original sample of researchsubjects who remained alive (y axis) at various time points (x axis)after the blood draw: a, subjects who were age 60-74 years at the timeof the blood draw; b, subjects who were age 75 years or older at blooddraw. In each panel “longer” identifies individuals from the top half ofthe TL distribution, and “shorter” identifies individuals from thebottom half of the distribution.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for predicting the survival orthe mortality risk of a eukaryotic organism. Specifically, the methodrelates to determining the length of telomeres and correlating thetelomere length with mortality risk associated with telomere length in apopulation. The present invention further provides methods ofdetermining telomere length and correlating the length with occurrenceof diseases associated with age, such a cardiovascular disease (e.g.,hypertension, myocardial infarction, etc.), neoplastic diseases (e.g.,malignant tumors, sarcomas, etc.), neurodegenerative disorders, andincreased susceptibility to infectious agents. In a further aspect, thepresent invention provides methods to determine the biological age, asopposed to chronological age, to provide a marker for correlating thebiological age with disease susceptibility and mortality risk.

The method of the present invention is useful in a variety ofapplications. Mortality or survival estimates based on telomere lengthgives a basis for identifying individuals or groups of individuals fortherapeutic intervention or medical screening. It is also useful inepidemiological studies for identifying environmental agents, forexample dietary factors, pathological agents, chemical toxins associatedwith telomere length and mortality. Similarly, the method may be used toidentify genetic factors, genetic linkage markers, or genes affectingtelomere length and age related diseases.

As discussed above, telomeres are specialized structures found at theends of linear chromosomes of eukaryotes. Telomeres are generallycomposed of short tandem repeats, with a repeat sequence unit specifiedby the telomerase enzyme particular to that organism. Telomere repeatsequences are known for a variety of organisms. For vertebrates, plants,certain types of molds, and some protozoans, the sequences are a perfectrepeats. For example, the human repeat sequence unit is (TTAGGG)_(n).(SEQ ID NO:5) In other organisms, the repeats sequences are irregular,such as those of Sacharomyces cerevisiae where the sequence is variableG1-3T/C1-3A. In some eukaryotic organisms, telomeres lack the shorttandem sequence repeats but have sequence elements that function astelomeres. For example, in the fruit fly Drosophila melanogaster, thetelomere is a composite of retrotransposon elements HeT-A and TART whilein the mosquito Anopheles gambiae the telomeres are arrays of complexsequence tandem repeats. For the purposes of the present invention,telomeres of different structures are encompassed within the scope ofthe present invention.

In addition to the repeat sequences, the 3′ end of some telomerescontains a single stranded region, which for humans is located on the Grich strand. The single strand is composed of (TTAGGG)_(n) (SEQ ID NO:5)repeats, with n being generally about 9-35, although it can be less ormore. As used herein, the length of the 3′ single stranded region canalso be correlated with mortality risk.

In one embodiment, telomere length may be determined for a singlechromosome in a cell. In another embodiment, the average telomere lengthor mean telomere length is measured for a single cell, and morepreferably for a population of cells. A change in telomere length is anincrease or decrease in telomere length, in particular an increase ordecrease in the average telomere length. The change may be relative to aparticular time point, i.e., telomere length of an organism at time t₁as compared to telomere length at some later time t₂. A change ordifference in telomere length may also be compared as against theaverage or mean telomere length of a particular cell population ororganismal population, preferably those members of a population notsuffering from a disease condition. In certain embodiments, change intelomere length is measured against a population existing at differenttime periods.

Although, telomere lengths may be determined for all eukaryotes, in apreferred embodiment, telemere lengths are determined for vertebrates,including without limitation, amphibians, birds, and mammals, forexample rodents, ungulates, and primates, particularly humans. Preferredare organisms in which longevity is a desirable trait or where longevityand susceptibility to disease are correlated. In another aspect, thetelomeres may be measured for cloned organisms in order to assess themortality risk or disease susceptibility associated with alteredtelomere integrity in these organisms.

Samples for measuring telomeres are made using methods well known in theart. The telomere containing samples may be obtained from any tissue ofany organism, including tissues of blood, brain, bone marrow, lymph,liver spleen, breast, and other tissues, including those obtained frombiopsy samples. Tissue and cells may be frozen or intact. The samplesmay also comprise bodily fluids, such as saliva, urine, feces,cerebrospinal fluid, semen, etc. Preferably, the tissue or cells arenon-stem cells, i.e., somatic cells since the telomeres of stem cellsgenerally do not decrease over time due to continued expression oftelomerase activity. However, in some embodiments, telomeres may bemeasured for stems cells in order to assess inherited telomerecharacteristics of an organism.

As used herein telomeric nucleic acids and other “target nucleic acids”or “target sequence” is meant a nucleic acid sequence on a double orsingle stranded nucleic acid. By “nucleic acid” or “oligonucleotide” orgrammatical equivalents herein is meant at least two nucleotidescovalently linked together. A nucleic acid of the present invention willgenerally contain phosphodiester bonds, although in some cases, nucleicacid analogs are included that may have alternate backbones, comprising,for example, phosphoramide (Beaucage, S. L. et al., Tetrahedron 49:1925-63 (1993), and references therein; Letsinger, R. L. et al., J. Org.Chem. 35: 3800-03 (1970); Sprinzl, M. et al., Eur. J. Biochem. 81:579-89 (1977); Letsinger, R. L. et al., Nucleic Acids Res. 14:3487-99(1986); Sawai et al, Chem. Lett. 805 (1984); Letsinger, R. L. et al., J.Am. Chem. Soc. 110: 4470 (1988); and Pauwels et al., Chemica Scripta26:141-49 (1986)), phosphorothioate (Mag, M. et al., Nucleic Acids Res.19:1437-41 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate(Briu et al., J. Am. Chem. Soc. 111:2321 (1989)),O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides andAnalogues: A Practical Approach, Oxford University Press, 1991), andpeptide nucleic acid backbones and linkages (Egholm, M., Am. Chem. Soc.114:1895-97 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992);Egholm, M., Nature 365: 566-68 (1993); Carlsson, C. et al., Nature 380:207 (1996), all of which are incorporated by reference). Other analognucleic acids include those with positive backbones (Dempcy, R. O. etal., Proc. Natl. Acad. Sci. USA 92:6097-101 (1995)); non-ionic backbones(U.S. Pat. Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141; and4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423(1991); Letsinger, R. L. et al., J. Am. Chem. Soc. 110: 4470 (1988);Letsinger, R. L. et al., Nucleoside & Nucleotide 13: 1597 (1994);Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modificationsin Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker etal., Bioorganic & Medicinal Chem. Lett. 4: 395 (1994); Jeffs et al., J.Biomolecular NMR 34:17 (1994)) and non-ribose backbones, including thosedescribed in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and7, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containingone or more carbocyclic sugars are also included within the definitionof nucleic acids (see Jenkins et al., Chem. Soc. Rev. 169-176 (1995));all references are hereby expressly incorporated by reference.

Telomeric nucleic acids, or a target nucleic acid, may be any length,with the understanding that longer sequences are more specific. In someembodiments, it may be desirable to fragment or cleave the samplenucleic acid into fragments of 100-10,000 base pairs, with fragments ofroughly 500 basepairs being preferred in some embodiments. Fragmentationor cleavage may be done in any number of ways well known to thoseskilled in the art, including mechanical, chemical, and enzymaticmethods. Thus, the nucleic acids may be subjected to sonication, Frenchpress, shearing, or treated with nucleases (e.g., DNase, restrictionenzymes, RNase etc.), or chemical cleavage agents (e.g.,acid/piperidine, hydrazine/piperidine, iron-EDTA complexes,1,10-phenanthroline-copper complexes, etc.).

The samples containing telomere and target nuclei acids may be preparedusing well-known techniques. For instance, the sample may be treatedusing detergents, sonication, electroporation, denaturants, etc., todisrupt the cells. The target nucleic acids may be purified as needed.Components of the reaction may be added simultaneously, or sequentially,in any order as outlined below. In addition, a variety of agents may beadded to the reaction to facilitate optimal hybridization,amplification, and detection. These include salts, buffers, neutralproteins, detergents, etc. Other agents may be added to improveefficiency of the reaction, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, etc., depending on the samplepreparation methods and purity of the target nucleic acid. When thetelomere nucleic acid is in the form of RNA, these nucleic acids may beconverted to DNA, for example by treatment with reverse transcriptase(e.g., MoMuLV reverse transcriptase, Tth reverse transcriptase, etc.),as is well known in the art.

Numerous methods are available for determining telomere length. In oneembodiment, telomere length is determined by measuring the mean lengthof a terminal restriction fragment (TRF). The TRF is defined as thelength—in general the average length—of fragments resulting fromcomplete digestion of genomic DNA with a restriction enzyme that doesnot cleave the nucleic acid within the telomeric sequence. Typically,the DNA is digested with restriction enzymes that cleaves frequentlywithin genomic DNA but does not cleave within telomere sequences.Typically, the restriction enzymes have a four base recognition sequence(e.g., AluI, HinfI, RsaI, and Sau3A1) and are used either alone or incombination. The resulting terminal restriction fragment contains bothtelomeric repeats and subtelomeric DNA. As used herein, subtelomeric DNAare DNA sequences adjacent to tandem repeats of telomeric sequences andcontain telomere repeat sequences interspersed with variabletelomeric-like sequences. The digested DNA is separated byelectrophoresis and blotted onto a support, such as a membrane. Thefragments containing telomere sequences are detected by hybridizing aprobe, i.e., labeled repeat sequences, to the membrane. Uponvisualization of the telomere containing fragments, the mean lengths ofterminal restriction fragments can be calculated (Harley, C. B. et al.,Nature. 345(6274):458-60 (1990), hereby incorporated by reference). TRFestimation by Southern blotting gives a distribution of telomere lengthin the cells or tissue, and thus the mean telomere length of all cells.

For the various methods described herein, a variety of hybridizationconditions may be used, including high, moderate, and low stringencyconditions (see, e.g., Sambrook, J. Molecular Cloning: A LaboratoryManual, 3rd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (2001); Ausubel, F. M. et al., Current Protocols inMolecular Biology, John Wiley & Sons (updates to 2002); herebyincorporated by reference). Stringency conditions are sequence-dependentand will be different in different circumstances, including the lengthof probe or primer, number of mismatches, G/C content, and ionicstrength. A guide to hybridization of nucleic acids is provided inTijssen, P. “Overview of Princples of Hybridization and the Strategy ofNucleic Acid Assays,” in Laboratory Techniques in Biochemistry andMolecular Biology: Hybridization with Nucleic Acid Probes, Vol 24,Elsevier Publishers, Amsterdam (1993). Generally, stringent conditionsare selected to be about 5-10° C. lower than the thermal melting point(i.e., T_(m)) for a specific hybrid at a defined temperature under adefined solution condition at which 50% of the probe or primer ishybridized to the target nucleic acid at equilibrium. Since the degreeof stringency is generally determined by the difference in thehybridization temperature and the T_(m), a particular degree ofstringency may be maintained despite changes in solution condition ofhybridization as long as the difference in temperature from T_(m) ismaintained. The hybridization conditions may also vary with the type ofnucleic acid backbone, for example ribonucleic acid or peptide nucleicacid backbone.

In another embodiment, telomere length is measured by quantitativefluorescent in situ hybridization (Q-FISH). In this method, cells arefixed and hybridized with a probe conjugated to a fluorescent label, forexample, Cy-3, fluoresceine, rhodamine, etc. Probes for this method areoligonucleotides designed to hybridize specifically to telomeresequences. Generally, the probes are 8 or more nucleotides in length,preferably 12-20 more nucleotides in length. In one aspect, the probesare oligonucleotides comprising naturally occurring nucleotides. In apreferred embodiment, the probe is a peptide nucleic acid, which has ahigher T_(m) than analogous natural sequences, and thus permits use ofmore stringent hybridization conditions. Generally, cells are treatedwith an agent, such as colcemid, to induce cell cycle arrest atmetaphase provide metaphase chromosomes for hybridization and analysis.Digital images of intact metaphase chromosomes are acquired and thefluorescence intensity of probes hybridized to telomeres quantitated.This permits measurment of telomere length of individual chromosomes, inaddition to average telomere length in a cell, and avoids problemsassociated with the presence of subtelomeric DNA (Zjilmans, J. M. etal., Proc. Natl. Acad Sci. USA 94:7423-7428 (1997); Blasco, M. A. etal., Cell 91:25-34 (1997); incorporated by reference).

In another embodiment, telomere lengths are measured by flow cytometry(Hultdin, M. et al., Nucleic Acids Res. 26: 3651-3656 (1998); Rufer, N.et al., Nat. Biotechnol. 16:743-747 (1998); incorporated herein byreference). Flow cytometry methods are variations of FISH techniques. Ifthe starting material is tissue, a cell suspension is made, generally bymechanical separation and/or treatment with proteases. Cells are fixedwith a fixative and hybridized with a telomere sequence specific probe,preferably a PNA probe, labeled with a fluorescent label. Followinghybridization, cell are washed and then analyzed by FACS. Fluorescencesignal is measured for cells in G_(O)/G₁ following appropriatesubtraction for background fluorescence. This technique is suitable forrapid estimation of telomere length for large numbers of samples.Similar to TRF, telemere length is the average length of telomereswithin the cell.

In a preferred embodiment, telomere lengths are determined by assessingthe average telomere length using polymerase chain reaction (PCR).Procedures for PCR are widely used and well known (see for example, U.S.Pat. Nos. 4,683,195 and 4,683,202). In brief, a target nucleic acid isincubated in the presence of primers, which hybridizes to the targetnucleic acid. When the target nucleic acid is double stranded, they arefirst denatured to generate a first single strand and a second singlestrand so as to allow hybridization of the primers. Any number ofdenaturation techniques may be used, such as temperature, although pHchanges, denaturants, and other techniques may be applied as appropriateto the nature of the double stranded nucleic acid. A DNA polymerase isused to extend the hybridized primer, thus generating a new copy of thetarget nucleic acid. The synthesized duplex is denatured and thehybridization and extension steps repeated. Carrying out theamplification in the presence of a single primer results inamplification of the target nucleic acid in a linear manner. For thepurposes of the present invention, linear amplification using a singleprimer is encompassed within the meaning of PCR. By reiterating thesteps of denaturation, annealing, and extension in the presence of asecond primer that hybridizes to the complementary target strand, thetarget nucleic acid encompassed by the two primers is amplifiedexponentially.

By “primer”, “primer nucleic acid”, “oligonucleotide primer”,“oligonucleotide probe” or grammatical equivalents as used herein ismeant a nucleic acid that will hybridize to some portion of the targetnucleic acid. The primers or probes of the present invention aredesigned to be substantially complementary to a target sequence suchthat hybridization of the target sequence and the primers of the presentinvention occurs, and proper 3′ base pairing allows primer extension totake place. Such complementarity need not be perfect. Thus, by“complementary” or “substantially complementary” herein is meant thatthe probes are sufficiently complementary to the target sequences tohybridize under normal reaction conditions. Deviations from perfectcomplementary are permissible so long as deviations are not sufficientto completely preclude hybridization. However, if the number ofalterations or mutations is sufficient such that no hybridization canoccur under the least stringent of hybridization conditions, as definedbelow, the sequence is not a complementary target sequence.

Although primers are generally single stranded, the nucleic acids asdescribed herein may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, RNA, or hybrid, wherethe nucleic acid contains any combination of deoxyribo- andribonucleotides, and any combination of bases, including uracil,adenine, thymine, cytosine, guanine, xanthine hypoxanthine, isocytosine,isoguanine, inosine, etc., although generally occurring bases arepreferred. As used herein, the term “nucleoside” includes nucleotides aswell as nucleoside and nucleotide analogs, and modified nucleosides suchas amino modified nucleosides. In addition, “nucleoside” includesnon-naturally occurring analog structures. Thus, for example, theindividual units of a peptide nucleic acid, each containing a base, arereferred herein as a nucleotide.

The size of the primer nucleic acid may vary, as will be appreciated bythose in the art, in general varying from 5 to 500 nucleotides inlength, with primers of between 10 and 100 being preferred, between 12and 75 being particularly preferred, and from 15 to 50 being especiallypreferred, depending on the use, required specificity, and theamplification technique.

For any primer pair, the ability of the primers to hybridize to eachother may be examined by aligning the sequence of the first primer tothe second primer. The stability of the hybrids, especially the thermalmelting temperature (T_(m)), may be determined by the methods describedbelow and by methods well known in the art. These include, but are notlimited to, nearest-neighbor thermodynamic calculations (Breslauer, T.et al., Proc. Natl. Acad. Sci. USA 83:8893-97 (1986); Wetmur, J. G.,Crit. Rev. Biochem. Mol. Biol. 26:227-59 (1991); Rychlik, W. et al., J.NIH Res. 6:78 (1994)), Wallace Rule estimations (Suggs, S. V. et al “Useof Synthetic oligodeoxribonucleotides for the isolation of specificcloned DNA sequences,” Developmental biology using purified genes, D. B.Brown, ed., pp 683-693, Academic Press, New York (1981), and T_(m)estimations based on Bolton and McCarthy (see Baldino, F. J. et al.,Methods Enzymol. 168: 761-77 (1989); Sambrook, J. et al., MolecularCloning: A Laboratory Manual, Chapter 10, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., (2001)). All references are herebyexpressly incorporated by reference. The effect of various parameters,including, but not limited to, ionic strength, probe length, G/Ccontent, and mismatches are taken into consideration when assessinghybrid stability. Consideration of these factors are well known to thoseskilled in the art (see, e.g., Sambrook, J., supra).

In a preferred embodiment, the primers of the present invention aredesigned to have similar T_(m)s. As used herein, primers with similarT_(m)s have a T_(m) difference of about 10° C. or less, preferably 5° C.or less, and more preferably 2° C. or less. Use of primer sets (e.g.,primer pairs) with similar or identical T_(m)s allow use of anannealing/extension temperature optimal for both primers and providessimilar amplification efficiency at a particular amplificationcondition. Advantages are the ability to use similar concentrations ofprimers, particularly at lower concentrations, which limits generationof unwanted amplification products. By comparison, when T_(m)s of theprimers are dissimilar, one primer is used at a higher concentration tocompensate for differences in amplification efficiency. This higherprimer concentration results in undesirable amplification products at alower number of PCR cycles.

In one aspect, primers with similar T_(m)s are made by altering thelength of primers or by selecting primers having similarguanosine-cytosine (GC) content. T_(m)s are assessed by the methodsdescribed above. As used herein, a “similar GC content” is meant aprimer set which has a GC content difference of about 10% or less, morepreferably a difference of about 5% or less, and most preferably adifference of about 2% or less, such that the primers display similarT_(m)s, as given above. In the primer design process, T_(m) and/or GCcontent are initially assessed for the region that hybridizes to thetarget nucleic acid. For primers with a non-hybridizing 5′ terminalregion described above, additional analysis of the T_(m) and GC contentis conducted for the entire primer sequence. Generally, primers aredesigned to have higher similarities of GC content at the 3′ terminalregion since it is this region that is extended by the polymerase.

The primers of the present invention may be used to amplify varioustarget nucleic acids. A single primer set, for example a primer pair,may be used to amplify a single target nucleic acid. In anotherembodiment, multiple primer sets may be used to amplify a plurality oftarget nucleic acids. Amplifications may be conducted separately foreach unique primer set, or in a single reaction vessel usingcombinations of primer sets, generally known in the art as multiplexing.When multiple primer sets are used in a single reaction, primers aredesigned to limit formation of undesirable products and limitinterference between primers of each primer set.

In one embodiment of a PCR based technique for measuring telomerelength, the method involves digesting genomic DNA with a restrictionenzyme to generate a DNA fragment containing the subtelomeric andtelomeric region. An oligonucleotide in the form of a linker iscovalently attached to the telomere end of the DNA fragment.Hybridization of a primer complementary to the linker followed by primerextension with a polymerase results in an extension product encompassingthe subtelomeric and telomeric region. Use of a second primer specificto the subtelomeric region allows for amplification of a product definedby the region between the two primers (see, e.g., U.S. Pat. No.5,834,193; incorporated by reference). In one aspect, the genomic DNA istreated with a single stranded specific nuclease to generate blunt ends,which allows efficient ligation of a double stranded linker onto the endof the telomere region. Subsequent amplification will provide arepresentative measure of telomere length even though treatment withnuclease will remove the 3′ single stranded region present at the end ofthe telomere.

In a variation on the primer extension method, an oligonucleotide primerspecific to the subtelomeric region is used to prime copying of thetelomeric and the subtelomeric region without attaching a linker to thetelomere end. Repeated rounds of hybridization, extension anddenaturation results in linear amplification of thesubtelomeric/telomeric region. The amplified product may be detected byusing labeled primers in the amplification reaction or by hybridizing alabeled probe to the amplified product.

Another method for determining telomere length by PCR involves use oftelomere specific primers designed to amplify the tandem repetitivetelomere sequences (Cawthon, R. M. Nucleic Acids Res. 30: e47 (2002); WO03/064615; incorporated by reference). This method involves uses ofprimers substantially complementary to telomere repeats but which do notundergo self-priming when the primers hybridize to each other during thePCR reaction. Amplfication by this PCR technique results in directamplification of telomere sequences, which when quantitated, provides ameasure of the average telomere length.

As used herein, a “repetitive unit”, “repeat unit”, “repetitive element”is meant a minimal nucleotide sequence which is reiterated or repeatedin the repetitive region, such as a telomere repeat sequence. In thepresent invention, the repetitive unit for amplification may compriserepetitive units of 1 or more nucleotides, more preferably repetitiveunits between 3 and 100 nucleotides, and most preferably repetitiveunits between 4 and 30 nucleotides. In general, these repetitive unitsare arranged in tandem fashion, although there may be non-repetitivenucleotides present between the repetitive units. By a “plurality” ofrepetitive elements herein is meant at least two or more repetitiveunits in the repetitive region. The number of repetitive units amplifiedfor each set of primers will depend on the length of the primer and thenucleotide length of the repetitive unit. As will be appreciated bythose skilled in the art, primer sequences and primer lengths may bechosen based on stability and specificity of the primer for therepetitive units.

Generally, the primers for direct amplification of telomere repeatscomprises a first primer which hybridizes to a first single strand ofthe target nucleic acid and a second primer which hybridizes to a secondsingle strand of the target nucleic acid, where the first and secondstrands are substantially complementary. The primers are capable ofprimer extension by polymerase when hybridized to their respectivestrands. That is, the primers hybridized to the target nucleic acid havetheir 3′ terminal nucleotide residues complementary to the nucleotideresidue on the target nucleic acid such that the primers are extendableby polymerase. The selected primers are complementary to repetitiveunits of the repetitive region. In one aspect, at least one nucleotideresidue of at least one of the primers is altered to produce mismatcheswith a nucleotide residue of at least one repetitive unit to which theprimer hybridizes, wherein the altered nucleotide residue also producesa mismatch with the 3′ terminal nucleotide residue of the other primerwhen the primers hybridize to each other. The inclusion of a mismatchprevents or limits primer extension of primer-primer hybrids.

In one preferred embodiment, at least one nucleotide residue of thefirst primer is altered to produce a mismatch between the alteredresidue and a nucleotide residue of at least one repetitive unit of thefirst strand to which the primer hybridizes, wherein the alterednucleotide residue also produces a mismatch with the 3′ terminalnucleotide residue of the second primer when the first and secondprimers hybridize to each other.

The altered nucleotide residue is preferably at least 1 nucleotideresidue, more preferably at least 2 nucleotide residues, and mostpreferably at least 3 nucleotide residues from the 3′ terminalnucleotide to allow efficient extension by polymerase when the alteredprimer hybridizes to target nucleic acids.

In another aspect, both primers used for directly amplifying repetitiveunits comprise at least one altered nucleotide residue such thathybridization of primers to each other generates mismatches between thealtered residues and the 3′ terminal residues of both primers. Thus, ina preferred embodiment, in addition to the altered nucleotide residue onthe first primer described above, at least one nucleotide residue of thesecond primer is altered to produce a mismatch between the alteredresidue and a nucleotide residue of at least one repetitive unit of thesecond strand to which the second primer hybridizes, wherein the alteredresidue on the second primer also produces a mismatch with the 3′terminal nucleotide of the first primer when the primers hybridize toeach other.

In yet another embodiment for amplifying repetitive units of arepetitive region, the present invention comprises a first primer whichhybridizes to more than one repetitive unit on a first single strand ofa target nucleic acid, and a second primer which hybridizes to more thanone repetitive unit on a second single strand of the target nucleicacid, where the first and second strands are substantiallycomplementary. The primers are capable of primer extension whenhybridized to their respective strands of the target nucleic acid, asdescribed above. In one aspect, nucleotide residues of at least one ofthe primers are altered to produce mismatches between the alteredresidues and the nucleotide residues at the identical nucleotideposition of each repetitive unit of the single strand of the targetnucleic acid to which the primer hybridizes. These altered nucleotideresidues also produce mismatches with the 3′ terminal nucleotide residueof the other primer when the primers hybridize to each other, thusfurther limiting primer-extension of primer-primer hybrids.

Accordingly, in one preferred embodiment, nucleotide residues of thefirst primer are altered to produce mismatches between the alteredresidues and nucleotide residues at the identical nucleotide position ofeach repetitive unit of the first strand of the target nucleic acid towhich the primer hybridizes. These altered nucleotides also producemismatches with the 3′ terminal nucleotide residue of the second primerwhen first second primer hybridizes to each other.

The altered nucleotide residues are preferably at least 1 nucleotideresidue, more preferably at least 2 nucleotide residues, and mostpreferably at least 3 nucleotide residues from the 3′ terminalnucleotide to allow efficient extension by polymerase when the alteredprimer hybridizes to target nucleic acids.

In another aspect, both primers comprise altered nucleotide residuessuch that hybridization of primers to each other results in mismatchesof the 3′ terminal nucleotide of both primers. Thus, in a preferredembodiment, in addition to the altered nucleotide residues on the firstprimer, nucleotide residues on the second primer are altered to producemismatches between the altered residues of the second primer andnucleotide residues at the identical nucleotide position of eachrepetitive unit of the second strand to which the primer hybridizes.These altered nucleotides of the second primer also produce mismatcheswith the 3′ terminal nucleotide residue of the first primer when theprimers hybridize to each other.

Since primers hybridized to target nucleic acids must be capable ofprimer extension, alterations of the first and second primers must be onnon-complementary nucleotides of the repetitive unit. Thus, in oneaspect, when both the first and second primers comprise alteredresidues, the alterations are at adjacent nucleotide positions of therepetitive unit. In another aspect, the alterations are situated onnon-adjacent nucleotide positions of the repetitive unit. In general,mismatches at adjacent nucleotide positions provide for the most numberof base paired or complementary residues between the altered nucleotideand the 3′ terminal nucleotide, which may be important for efficientlyamplifying short repetitive sequences (i.e., 3-6 basepairs).

In another embodiment, the first and second primers further comprise a5′ terminal region that does not hybridize (i.e., basepair) with thetarget nucleic acid. The unpaired region comprises one or morenucleotides, with a preferred range of 3 to 60 nucleotides, and a mostpreferred range of 4 to 30 nucleotides. When the primers are directedtowards amplification of repetitive units of a repetitive region, the 5′unpaired region blocks the 3′ ends of the replicated primer extensionproducts from initiating nucleic acid synthesis from the internalrepetitive units of the amplification products during subsequentamplification cycles.

Although the 5′-terminal unpaired region may be of any sequence whichdoes not hybridize to the target nucleic acid, in a preferredembodiment, the unpaired region comprises restriction sites, uniquesequences for purposes of sequencing or primer extension reactions(i.e., amplification), or tag sequences for detecting and measuring theamplified product.

As discussed above, in a preferred embodiment, the primers of thepresent invention are designed to have similar T_(m)s to limitgeneration of undesirable amplification products and to permitamplification and detection of several target nucleic acids in a singlereaction volume. In addition, since the telomeres of various organismshave differing repetitive unit sequences, amplifying telomeres of aspecific organism will employ primers specific to the repetitive unit ofeach different organism. Human telomeric sequences are used herein toillustrate practice of the present invention for direct amplificationand quantitation of tandemly repeated nucleic acid sequences, but is notlimited to the disclosed specific embodiment.

Amplification reactions are carried out according to procedures wellknown in the art, as discussed above (see, e.g., U.S. Pat. Nos.4,683,195 and 4,683,202). The time and temperature of the primerextension step will depend on the polymerase, length of target nucleicacid being amplified, and primer sequence employed for theamplification. The number of reiterative steps required to sufficientlyamplify the target nucleic acid will depend on the efficiency ofamplification for each cycle and the starting copy number of the targetnucleic acid. As is well known in the art, these parameters can beadjusted by the skilled artisan to effectuate a desired level ofamplification. Those skilled in the art will understand that the presentinvention is not limited by variations in times, temperatures, bufferconditions, and the amplification cycles applied in the amplificationprocess.

In hybridizing the primers to the target nucleic acids and in theamplification reactions, the assays are generally done under stringencyconditions that allow formation of the hybrids in the presence of targetnucleic acid. Those skilled in the art can alter the parameters oftemperature, salt concentration, pH, organic solvent, chaotropic agents,or other variables to control the stringency of hybridization and alsominimize hybridization of primers to non-specific targets (i.e., by useof “hot start” PCR or “touchdown” PCR).

Following contacting the primers to the target nucleic acids, thereaction is treated with an amplification enzyme, generally apolymerase. A variety of suitable polymerases are well known in the art,including Taq polymerase, KlenTaq, Tfl polymerase, DynaZyme etc.Generally, although all polymerases are applicable to the presentinvention, preferred polymerases are thermostable polymerases lacking 3′to 5′ exonuclease activity since use of polymerases with strong 3′ to 5′exonuclease activity tends to remove the mismatched 3′ terminalnucleotides. Also useful are polymerases engineered to have reduced ornon-functional 3′ to 5′ exonuclease activities (e.g., Pfu(exo-),Vent(exo-), Pyra(exo-), etc.). Also applicable are mixtures ofpolymerases used to optimally extend hybridized primers. In anotheraspect, polymerase enzymes useful for the present invention areformulated to become active only at temperatures suitable foramplification. Presence of polymerase inhibiting antibodies, whichbecome inactivated at amplification temperatures, or sequestering theenzymes in a form rendering it unavailable until amplificationtemperatures are reached, are all suitable. These polymeraseformulations allow mixing all components while preventing priming ofnon-target nucleic acid sequences.

In another aspect, those skilled in the art will appreciate that variousagents may be added to the reaction to increase processivity of thepolymerase, stabilize the polymerase from inactivation, decreasenon-specific hybridization of the primers, or increase efficiency ofreplication. Such additives include, but are not limited to, dimethylsulfoxide, formamide, acetamide, glycerol, polyethylene glycol, orproteinacious agents such as E. coli. single stranded DNA bindingprotein, T4 gene 32 protein, bovine serum albumin, gelatin etc. Inanother aspect, the person skilled in the art can use various nucleotideanalogs for amplification of particular types of sequences, for exampleGC rich or repeating sequences. These analogs include c⁷-dGTP,hydroxymethyl-dUTP, dITP, 7-deaza-dGTP etc.

The products of the amplification are detected and analyzed by methodswell known to those skilled in the art. Amplified products may beanalyzed following separation and/or purification of the products, or bydirect measurement of product formed in the amplification reaction.Separation and purification methods include, among others,electrophoresis (i.e., agarose or acrylamide gels), chromatography(i.e., affinity, molecular sieve, reverse phase etc.) and hybridization.The purified products may be subjected to further amplifications as iswell known in the art. For detection, the product may be identifiedindirectly with fluorescent compounds, for example with ethidium bromideor SYBR™Green or by hybridization with labeled nucleic acid probes.Alternatively, labeled primers or labeled nucleotides are used in theamplification reaction to label the amplification product. The labelcomprises any detectable moiety, including fluorescent labels,radioactive labels, electronic labels, and indirect labels such asbiotin or digoxigenin. When indirect labels are used, a secondarybinding agent that binds the indirect label is used to detect thepresence of the amplification product. These secondary binding agentsmay comprise antibodies, haptens, or other binding partners (e.g.,avidin) that bind to the indirect labels. Secondary binding agents arepreferably labeled with fluorescent moieties, radioactive moieties,enzymes etc.

In one embodiment, the amplification product may be detected andquantitated during the amplification reaction by real time quantitativePCR, variations of which are well known in the art. For instance, theTaqMan system uses a probe primer which hybridizes to sequences in aninternal sequence within the nucleic acid segment encompassed by theprimers used to amplify the target nucleic acid (Heid, C. A. et al.,Genome Res. 6:986-94 (1996); Holland, P. M. et al., Proc. Natl. Acad.Sci. USA 88: 7276-80 (1991)). This probe is labeled with two differentfluorescent dyes (i.e., dual-labeled flurogenic oligonucleotide probe),the 5′ terminus reporter dye (TAMRA) and the 3′ terminus fluorescencequenching dye (FAM). Cleavage of the probe by the 5′ to 3′ exonucleaseactivity of DNA polymerase during the extension phase of PCR releasesthe flurogenic molecule from proximity of the quencher, thus resultingin increased fluorescence intensity.

In another aspect, real time quantitative PCR may be based onfluorescence resonance energy transfer (FRET) between hybridizationprobes (Wittwer, C. T., Biotechniques 22:130-138 (1997)). In thismethod, two oligonucleotide probes hybridize to adjacent regions of thetarget nucleic acid sequence. The upstream probe is labeled at the 3′terminus with an excitor dye (i.e., FITC) while the adjacentlyhybridizing downstream probe is labeled at the 5′ terminus with areporter dye. Hybridization of the two probes to the amplified targetnucleic acid sequences positions the two dyes in close spatial proximitysufficient for FRET to occur. This allows monitoring the quantity ofamplified product during the polymerase chain reaction. A similarapproach is used in the molecular beacon probes (Tyagi, S., Nat.Biotechnol. 16: 49-53 (1998)). Molecular beacons are oligonucleotideprobes comprising a quencher dye and a reporter dye at the opposite endsof the PCR product specific oligonucleotide. The dyes may also functionbased on FRET, and therefore may also be comprised of an excitation dyeand a reporter dye. Short complementary segments at the 5′ and 3′terminal regions allow for formation of a stem-loop structure, whichpositions the dyes at the terminal ends of the oligonucleotide intoclose proximity, thus resulting in fluorescence quenching or FRET. Whenthe oligonucleotide hybridizes to a PCR product through complementarysequences in the internal region of the molecular beacon probe,fluorescence of the oligonucleotide probe is affected, thus allowingmonitoring of product synthesis.

In a preferred embodiment, real time quantitative PCR may usefluorescent dyes that preferentially bind to double stranded nucleicacid amplification products during the PCR reaction to permit continuousmonitoring of product synthesis (see, e.g., Higuchi, R. et al.,Biotechnology 11: 1026-30 (1993); T. B. et al., Biotechniques 24: 954-62(1998)). Suitable fluorescent dyes include, among others, ethidiumbromide, YO PRO-1™ (Ishiguro, T., Anal. Biochem. 229: 207-13 (1995)),and SYBR™ Green dyes (Molecular Probes, Eugene, Oreg., USA). Whenamplifying target nucleic acids comprising repetitive regions, FRET ormolecular beacon based probes are not preferred if FRET or molecularbeacon probes are directed to repetitive units since they will hybridizeto repetitive sequences on the primers, thereby failing to distinguishbetween primers and amplified product.

In a further preferred embodiment, real time quantitative PCR isaccomplished with primers containing a single flurophore attached nearthe 3′ terminal nucleotide (Nazarenko, I. et al., Nucleic Acids Res. 30:e37 (2002); Nazarenko, I. et al., Nucleic Acids Res. 30: 2089-2195(2002); LUX™ Fluorogenic Primers, Invitrogen, Palo Alto, Calif.; herebyincorporated by reference). The 5′ end of these primers have a 5 to 7nucleotide extension capable of hybridizing to the 3′ terminal region togenerate a blunt-ended hairpin (i.e., stem-loop) structure, whoseformation results in fluorescence quenching of the fluorophore. When theprimer forms a duplex, for example by primer extension on a template,the quenching is reduced or eliminated, thus providing a measure of PCRproduct in the sample. Because only a single flurophore is used,different flurophores may be used and detected in a single reaction.Consequently, these primers are useful for amplification and detectionof a plurality of different target nucleic acids in a single reactionvessel by use of different primer sets with distinguishable flurophores.As discussed herein, various target nucleic acids include combinationsof single copy genes and repetitive sequences, as further describedherein.

Instrumentation suitable for real time monitoring of PCR reactions isavailable for use in quantitative PCR methods (ABI Prism 7700, AppliedBiosystems Division, Perkin Elmer, Fosters City, Calif., USA;LightCycler™, Roche Molecular Biochemicals, Indianapolis, Ind., USA).Other real time PCR detection systems are known to those of ordinaryskill in the art.

When real time quantitative PCR is used to detect and measure theamplification products, various algorithms are used to calculate thenumber of target nucleic acids in the samples (see, e.g., ABI Prism 7700Software Version 1.7; Lightcycler™ Software Version 3). Quantitation mayinvolve use of standard samples with known copy number of the targetnucleic acid, and generation of standard curves from the logarithms ofthe standards and the cycle of threshold (C_(t)). In general, C_(t) isthe PCR cycle or fractional PCR cycle where the fluorescence generatedby the amplification product is several deviations above the baselinefluorescence (Higuchi, R. et al., supra). Real time quantitative PCRprovides a linearity of about 7 to 8 orders of magnitude, which allowsmeasurement of the copy number of target nucleic acids over a widedynamic range. The absolute number of target nucleic acid copies can bederived from comparing the C_(t) values of the standard curve and thesamples.

The copy number of telomere repeats or target nucleic acid may also bedetermined by comparative quantitative real time PCR. Use of nucleicacids of known copy number or consistent copy number allows quantitationof the copy number of target nucleic acids in a sample. The standard maybe a single copy gene, a nucleic acid of known copy number, or whenquantitating RNA copy number, a constitutively expressed housekeepinggene (see Johnson, M. R. Anal. Biochem. 278: 175-84 (2000); Boulay,J.-L., et al., Biotechniques 27: 228-32 (1999)).

The amplified products are quantitated as described above. In apreferred embodiment, real time quantitative PCR is used to determinethe copy number of the telomere repetitive units in the target nucleicacid sample. Standards for determining and comparing telomere repetitiveunit number include use of single copy genes (e.g., ribosomalphosphoprotein 364B) or a target nucleic acid of known copy number(e.g., a plasmid with known number of telomere repetitive units). By themethods described herein, the copy number of repetitive units of a largenumber of samples may be quantitated for purposes of determining thenumber of telomere repetitive units, and thus the average length oftelomeres.

Following determination of telomere length for an individual orpopulation, the measured values may correlated with mortality risk ordisease occurrence for a population, and in particular, individualswithin a population. A variety of statistical analysis may be used forthis purpose, including, but not limited to, Cox proportional hazardregression analysis, Kaplan-Meier survival distribution estimate, PetoWilcoxon test, maximum likelihood analysis, multiple regressionanalysis, and others. Such statistical methods, including various formsof survival analysis, are well known in the art and described instandard works on statistics (see, e.g., Cantor, A. B. SAS SurvivalAnalysis Techniques for Medical Research, 2nd Ed., SAS Publishing, Cary,N.C. (2003); Hosmer, D. W. et al., Applied Survival Analysis: RegressionModeling of Time to Event Data Wiley-Interscience, Hoboken, N.J. (2002);Lee, E. T., Statistical Methods for Survival Data Analysis, 2nd Ed.,John Wiley & Sons, Hoboken, N.J. (1992); and Schork, M. A. andRemington, R. D., Statistics with Applications to the Biological andHealth Sciences, 3rd Ed., Prentice Hall, Upper Saddle River, N.J.(2000). All publications are incorporated by reference in theirentirety.

In the present invention, the telomere length, or in some embodiments,rates of changes in telomere length, is correlated with mortality risk.Thus, the telomere length is a useful marker for predicting survival ormortality risk for different age groups. In a further embodiment, themeasured telomere length is used for correlating occurrence of diseases,particularly age related diseases, including, but not limited tocardiovascular disease (e.g., hypertension, myocardial infarction,stroke, etc.), neoplastic diseases (e.g., cancers, carcinomas, malignanttumors, etc.), and susceptibility to infectious diseases.

The correlations between telomere length and mortality risk or diseaseoccurrence may be used for disease prognosis and therapeuticapplications. In one preferred embodiment, the method is used toidentify individuals or groups of individuals with telomeres lengthsfalling within the ranges of those populations with increased mortalityrisk, advanced biological age, or increased risk for death from agerelated diseases. This may provide a basis for identifying individualswho may warrant additional medical screening or early medicalintervention.

For example, it is shown herein that there is a statisticallysignificant increased mortality rate ratio for death from infectiousdisease for those in the bottom half for telomere length compared tothose in the top half for telomere length. In addition, a statisticallysignificant increase in mortality rate ratio for death from heartdisease is observed for those in the bottom half vs. those in the tophalf for telomere length. Thus, in clinical practice, the telomerelength assay results may help physicians in assessing each specificpatient's risk of these diseases, and so may make a difference in thephysician's treatment plan for the patient.

In another application, the method may be used to identify environmentalfactors affecting or related to telomere shortening. Populations withheightened exposure to carcinogens, teratogens, radiation, or otherenvironmental factors may be tested and examined for the existence ofany statistically significant correlations between exposure, telomerelength, and age related diseases. This may identify environmental riskfactors which can be reduced or minimized to decrease adverse effect onthe health of populations.

In a further application, the methods of the present invention may beused to identify genes, genetic linkages, or other genetic factorscontributing to telomere shortening and their relation to mortality andsusceptibility to various age related disease. Studies of largepopulations can facilitate identification of such risk factors.

The foregoing descriptions of specific embodiments of the presentinvention have been presented for purposes of illustration anddescription. They are not intended to be exhaustive or to limit theinvention to the precise forms disclosed, and obviously manymodifications and variations are possible in light of the aboveteachings.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

EXAMPLES Example 1 Determining Average Telomere Length

Genomic DNA was extracted from blood samples by standard procedures. Thesamples used to compare quantitative PCR vs. Southern blot approaches totelomere measurement were donated by 21 unrelated individuals (11 womenand 10 men, age range 61-94 years) from Utah families that are part ofthe Centre pour les Etudes du Polymorphisme Humaine (CEPH) collectionused worldwide for building the human genetic linkage map (White, R. etal. (1985) Nature 313: 101-105). Purified DNA samples were diluted in96-well microtiter source plates to approximately 1.75 ng/μl in 10 mMTris-HCl, 0.1 mM EDTA, pH 7.5 (final volume 300 ul per well), heated to95° C. for 5 minutes in a thermal cycler, quick-chilled by transfer toan ice-water bath for 5 minutes, centrifuged briefly at 700×g, sealedwith adhesive aluminum foil, and stored at 4° C. until the time ofassay.

Real time quantitative PCR on the extracted DNA samples are performed onseparate 96 well plates. Two master mixes of PCR reagents were prepared,one with the telomere (T) primer pair, the other with the single copygene (S) primer pair. Thirty microliters of T master mix was added toeach sample well and standard curve well of the first plate, and 30microliters of S master mix was added to each sample well and standardcurve well of the second plate. For each individual in whom the T/Sratio was assayed, three identical 20 ul aliquots of the DNA sample (35ng per aliquot) were added to plate 1, and another three aliquots wereadded to the same well positions in plate 2. For each standard curve,one standard DNA sample was diluted serially in TE (10 mM Tris, 1 mMEDTA, pH 7.0) by ˜1.68-fold per dilution to produce five concentrationsof DNA ranging from 0.63 ng/ul up to 5 ng/ul, which were thendistributed in 20 ul aliquots to the standard curve wells on each plate.The plates were then sealed with a transparent adhesive cover,centrifuged briefly at 700×g, and stored at 4° C. in the dark until thePCR was performed (0-3 days later).

The final concentrations of reagents in PCR with tel 1 and tel 2 primerswere 150 nM 6-ROX and 0.2×SYBR™ Green 1 (Molecular Probes, Inc.); 15 mMTris-HCl, pH 8.0; 50 mM KCl; 2 mM MgCl₂; 0.2 mM of each dNTP; 5 mM DTT;1% DMSO; and 1.25 units of AmpliTaq Gold DNA polymerase (AppliedBiosystems, Inc.). The thermal cycling profile began with a 95° C.incubation for 10 min. to activate the AmpliTaq Gold DNA polymerase.With tel 1 and tel 2 primers, there followed 18 cycles of 95° C.×15 s,54° C.×2 min. Telomere primer concentrations were tel 1, 270 nM; tel 2,900 nM. The primer sequences in 5′ to 3′ direction were tel 1,GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT; (SEQ ID NO:1)and tel 2,TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA (SEQ ID NO:2).

Alternatively, the primer used for amplifying the telomere repeatsequences were optimized to have similar T_(m)s, particularly bydesigning the primers to have similar or identical GC content: tel 1b,CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT (SEQ ID NO:6); and tel 2b,GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT (SEQ ID NO:7). Each of the T_(m)optimized primers has perfect complementarity to telomere DNA sequencesin the last five bases at the 3′ end of the primer. As with tel 1 andtel 2 primers, the optimized primers have a purposefully introducedsingle base substitution at the sixth base from the 3′ end and at everysixth base thereafter in the 5′ direction, for a total of fiveintroduced base changes. This results in five single base mismatcheswhen the primer is optimally hybridized to telomeric DNA. Hybridizationof the optimized primers to each other results in base pairing at fourout of the six positions in the repeat and where the 3′ terminal base ofeach primer is mismatched relative to the other primer.

For amplifications using tel 1b and tel 2b, the amplificationsconditions were 0.4× Sybr Green I, 1.5 mM MgCl₂, 1% DMSO, 2.5 mM DTT,200 micromolar each dNTP, 0.75 Units of AmpliTaq Gold DNA polymerase,450 nM of tel 1b primer, and 450 nM of tel 2b primer, in a final volumeof 30 microliters per reaction. The thermal cycling profile was 95°C.×10 min. to activate the polymerase; followed by 18 cycles of 95° C.for 15 sec. (denaturation) and 56° C. for 2 min. (anneal/extend). No ROXdye is needed for this assay.

For PCR with single copy gene 36B4 primers, reactions conditions were150 nM 6-ROX and 0.2× SYBR™ Green I (Molecular Probes, Inc.); 15 mMTris-HCl, pH 8.0; 50 mM KCl; 2 mM MgCl₂; 0.2 mM of each dNTP; 5 mM DTT;1% DMSO; and 1.25 units of AmpliTaq Gold DNA polymerase (AppliedBiosystems, Inc.). The final 36B4 (single copy gene) primerconcentrations were 36B4u, 300 nM and 36B4d, 500 nM. The thermal cyclingprofile began with a 95° C. incubation for 10 min. to activate theAmpliTaq Gold DNA polymerase. Subsequently, there followed 30 cycles of95° C.×15 s, 58° C.×1 min. Alternatively, the amplification conditionswere 0.4× Sybr Green I, 3.5 mM MgCl₂, 1% DMSO, 2.5 mM DTT, 200micromolar each dNTP, 0.75 Units of AmpliTaq Gold DNA polymerase, 300 nMof 36B4u primer, and 500 nM of 36B4d primer, in a final volume of 30microliters per reaction. The thermal cycling profile was 95° C.×10 min.to activate the polymerase; followed by 30 cycles of 95° C. for 15 sec.(denaturation) and 56° C. for 1 min. (anneal/extend). No ROX dye isneeded for this assay. Sequences of the single copy gene primers are36B4u, CAGCAAGTGGGAAGGTGTAATCC (SEQ ID NO:8); and 36B4d,CCCATTCTATCATCAACGGGTACAA (SEQ ID NO:9). (The 36B4 gene, which encodesacidic ribosomal phosphoprotein PO, is located on chromosome 12; seeBoulay et al. (1999) Biotechniques 27: 228-32).

All PCRs were performed on ABI Prism 7700 Sequence Detection System(Applied Biosytems, Inc., Foster City, Calif., USA), a thermal cyclerequipped to excite and read emissions from fluorescent molecules duringeach cycle of the PCR. ABI's SDS version 1.7 software was then used togenerate the standard curve for each plate and to determine the dilutionfactors of standard corresponding to the T and S amounts in each sample.

In the presence of 35 ng of human DNA, a telomere PCR product wasdetectable by real time quantitative PCR beginning from about 9 cyclesof PCR. Analysis of the product after 25 cycles by electrophoresis onagarose gels and staining with ethidium bromide shows a smear ofproducts beginning from about 76 base pairs, which is equivalent to thesum of the lengths of the telomere specific primers, to products ofabout 400 base pairs. The copy number of the PCR is proportional to thenumber of sites available for binding of the primer in the first cycleof the PCR. Omitting the genomic DNA results in no detectableamplification product after 25 cycles for either the telomere or singlecopy gene primers.

Mean telomere restriction fragment (TRF) lengths were determined asdescribed by Slagboom et al. (1994) Am. J. Hum. Genet 55: 876-82.Approximately 0.5 ug of purified whole blood DNA was digested tocompletion with Hae III restriction enzyme. Digested samples were thenmixed with DNA size standards, separated by electrophoresis on agarosegels, and transferred to a nylon membrane. The membranes were hybridizedwith ³²P end labeled oligonucleotide, (TTAGGG)₇ (SEQ ID NO:10), washedto remove non-specifically bound probe, exposed to a phosphor plate for1 to 5 days, and scanned with a PhosphorImager (Molecular Dynamics,Inc.). Blots were then stripped of the telomere probe, hybridized withradiolabeled probe for the DNA size standards, washed, exposed to aphosphor plate, and scanned. The size standard images and telomere smearimages were then superimposed to locate the positions of the sizeintervals within the telomere smears. Mean TRF length was thencalculated as mean TRF length=(S OD_(i))/(S OD_(i)/L_(i)), where OD_(i)is total radioactivity above background in interval i, and L_(i) is theaverage length of i in basepairs. This entire procedure was performedtwice; i.e., the two mean TRF length values determined on eachindividual were obtained from two independent experiments.

To measure the T/S value (telomere to single copy gene ratio), the C_(t)value—the fractional cycle number at which the amplification sample'saccumulating fluorescence crosses a set threshold value that is severalstandard deviations above background fluorescence—was determined forsamples amplified with telomere specific (T) primers and single copygene specific (S) primers. Since the amount of PCR product approximatelydoubles in each cycle of the PCR, the T/S ratio is approximately[2^(Ct(telomeres))/2^(Ct(single copy gene))]⁻¹=2^(−ΔCt). The averageΔC_(t) was −9.05 (see FIG. 2). That is, PCR of a single copy generequired about 9 more cycles than PCR of telomeres to produce equivalentfluorescent signal as measured by real time PCR. The standard deviationwas 1.48%.

The relative T/S ratio, which is the T/S of one sample relative to theT/S of another sample, is expressed as 2^(−(ΔCt1−ΔCt2))=2^(−ΔΔCt). Thisformula allows calculating the relative T/S ratio of each sample. DNAsamples of 21 unrelated patients were amplified and quantitated by realtime quantitative PCR (see Experiment 2). Comparison of the relative T/Sratio calculated from PCR correlated well with the mean TRF lengthsdetermined by Southern hybridization (see FIG. 3). The y intercept isabout 3.6 kbp, which is approximately the mean length of thesubtelomeric region between the restriction enzyme recognition sites andthe beginning of the telomeric hexamer repeats (Hultdin, M. (1998)Nucleic Acids. Res. 26: 3651-56). Moreover, the observed averagetelomere length in whole blood as measured by relative T/S ratio variesover a 2.5 range among unrelated age and sex-matched adults. This rangeof variability is in excellent agreement with other studies on the rangeof variation of TRF lengths in age matched adults if the averagesubtelomeric length of 3.4 kbp is subtracted from each reported mean TRFlength (Hultdin, M. (1998) Nucleic Acids Res. 3651-56; Vaziri, H. et al.(1993) Am. J. Hum. Genet. 52: 661-67).

Example 2 Telomere Length and Mortality in Humans

The 143 research subjects were unrelated Utah residents aged 60-97years, who donated blood from 1982-1986 to contribute to the CEPH(Centre d'Etude du Polymorphisme Humain) collection of cell lines usedto build the human genetic linkage map (White R. et al, Nature 313:101-105 (1985)) and for whom follow-up survival data were available.Birth and death dates were obtained from the Utah Population Database(UPDB), and from the Social Security Death Index. There were 101 knowndeaths by mid-2002. For the remaining 42 subjects, date at which theywere last known to be alive was established post blood draw. Thesurvival analysis by cause of death (from Utah death certificates codedto the International Classification of Diseases, 9th and 10th revisions)was restricted to the 124 individuals with UPDB identification numbers.This study was approved by the University of Utah's Institutional ReviewBoard.

Relative TLs in total genomic DNA prepared directly from the originalblood draws were measured by a quantitative polymerase chain reactionassay (Cawthon, R. M. Nucleic Acids Res. 30: e47 (2002)), whichdetermines the relative ratio of telomere repeat copy number to singlecopy gene copy number (T/S ratio) in experimental samples as compared toa reference DNA sample. In another set of DNA samples T/S ratios weremeasured relative to the same reference DNA sample, and mean terminalrestriction fragment (TRF) lengths were determined (Cawthon, supra). Theslope of the plot of mean TRF length vs. T/S for these samples served asthe conversion factor for calculating approximate telomere lengths inbasepairs for each T/S ratio in this survival study. In these subjectsaged 60-97 years, TL ranged from 1930-4310 bp. Each one year increase inage at blood draw was associated with a 0.0048 decrease in the relativeT/S ratio (95% CI [0.00137 to 0.00823], P=0.0074), corresponding toabout 14 bp of telomere sequence lost per year. Women and men did notdiffer significantly in the rate of telomere shortening estimated fromthese cross-sectional data (P=0.645). Women's telomeres were 3.5% longerthan those of men after adjusting for age, but this difference was notstatistically significant (P=0.157).

Cox proportional hazard regression models (Ghali, W. A. et al. JAMA 286:1494-1497 (2001)) were used to test whether differences in telomerelength (TL) among statistically age-matched individuals were associatedwith differences in survival. TL, when analyzed as a continuousvariable, was inversely associated with the age-adjusted mortality rate(Cox proportional hazards regression coefficient=−1.87, 95% CI [−3.35 to−0.392], P=0.013). In all other analyses TL was treated as a dichotomoustrait (“shorter” vs. “longer”), using all available samples in eachcomparison (i.e., bottom half of the TL distribution vs. top half, andbottom 25% vs. top 75%). Because older individuals tend to have shortertelomeres than younger individuals, the use of a single TL distributionfor the entire sample would result in a higher proportion of older thanyounger subjects being scored as “shorter” for TL, and a higherproportion of younger than older subjects being scored as “longer” forTL. To achieve more balanced proportions of subjects with “shorter” vs.“longer” TLs at every age, the sample was stratified into six categoriesof age at draw (60-64 years, number of subjects: n=19; 65-69, n=37;70-74, n=37;75-79, n=29; 80-84, n=12; and 85+, n=9), and the TLdistribution was determined independently within each category.Individuals in the bottom half for TL in each age group were pooledtogether, and their survival was compared to that of the pooled top halfindividuals. Similarly, individuals in the bottom quartile for TL ineach age group were pooled, and their survival was compared to that ofthe pooled top 75% individuals. There was no significant difference inthe mean age at draw between the compared groups (i.e., bottom vs. tophalf, bottom 25% vs. top 75%). Survival was assessed beginning with thetime at blood draw, except as noted. Cox models were used to control forvariation in mortality rate due to age differences, both between the ageat blood draw categories, and within each age group.

Individuals with shorter telomeres had a mortality rate nearly twicethat of individuals with longer telomeres (Table 1). The loss in mediansurvival associated with shorter telomeres (FIG. 1) was 4.8 years forwomen, and 4.0 years for men (averaged across all age-at-blood-drawcategories). TL was a significant predictor of mortality when measuredfrom ages 60 to 74 (P=0.021), and a moderate predictor when measuredfrom age 75 onward (P=0.086) (Table 1, FIG. 2). Excess mortality risksassociated with short vs. long telomeres did not vary by sex (P=0.878),age at blood draw (P=0.946), or time since blood draw (P=0.851). Theexcess mortality rates of those in the bottom half of TL remainedsignificant even when only those subjects surviving at least 5 yearsafter the blood draw were included in the analysis (P=0.0063, n=112).TABLE 1 Mortality rate ratios associated with having short vs. longtelomeres in whole blood DNA Mortality 95% Confidence P rate ratiointerval value All-cause mortality Both sexes combined (n = 143, 1.86(1.22-2.83) 0.004 d = 101) Women (n = 71, d = 46) 2.16 (1.07-4.39) 0.033Men (n = 72, d = 55) 1.94 (1.01-3.74) 0.047 Age at draw <75 (n = 93, d =53) 1.96 (1.11-3.48) 0.021 Age at draw ≧75 (n = 50, d = 48) 1.73(0.93-3.24) 0.086 Cause-specific mortality Heart (n = 124, d = 30) 3.18(1.36-7.45) 0.008 Cerebrovascular (n = 124, d = 15) 1.35 (0.36-5.13)0.660 Cancer (n = 124, d = 12) 1.43 (0.34-6.03) 0.625 Infectious (n =124, d = 8) 8.54 (1.52-47.9) 0.015 Other known (n = 124, d = 16) 2.15(0.71-6.50) 0.174 All known causes except 1.70 (0.82-3.53) 0.156 heart +infectious (n = 124, d = 43)

Each mortality rate ratio (MMR) presented here is the ratio of the deathrate for subjects with shorter telomeres to the death rate for subjectswith longer telomeres. In all five categories of all-cause mortality,and in the first category of cause-specific mortality (heart disease),the MMR reported above is for individuals from the bottom half of thetelomere length (TL) distribution vs. those from the top half of thedistribution. In the remaining five categories of cause-specificmortality, the MMR reported is for individuals from the bottom 25% ofthe TL distribution vs. those from the top 75% of the distribution. n:total number of individuals in each analysis. d: for all-causemortality, the number of deceased individuals in each analysis; forcause-specific mortality, the number of individuals in each analysis whodied from the listed cause of death.

The rate ratios for cause-specific mortality associated with havingshorter vs. longer telomeres are also presented in Table 1. Subjectsfrom the bottom half of the TL distribution had a heart diseasemortality rate that was over three times that of subjects from the tophalf. This elevated risk of dying from heart disease remainedsignificant even when the analysis was limited to subjects who survivedat least five years after the blood draw (number of heart diseasedeaths=21, mortality rate ratio 4.87, 95% CI [1.59 to 14.9], P=0.006).Mortality rates for cerebrovascular disease and cancer were also higherin individuals with shorter telomeres, although not significantlyhigher. The mortality rate from infectious disease was eight timeshigher for individuals in the bottom 25% of the TL distribution than forindividuals in the top 75%, a statistically significant difference.Among the infectious disease deaths, the shortest time between the blooddraw and death was 1.5 years. The remaining 16 deaths due to knowncauses, other than those mentioned above, were treated as a singlecategory of mortality; the risk of dying in this category was alsohigher in individuals with shorter telomeres, although not significantlyhigher.

1. A method for predicting survival of an organism, said methodcomprising: a) determining telomere length of said organism; and b)correlating said telomere length with mortality risk associated withtelomere length in a population of the organism.
 2. The method accordingto claim 1, wherein in said organism is human.
 3. The method accordingto claim 1, wherein telomere length is the average telomere length. 4.The method according to claim 3, wherein said average telomere length isdetermined by polymerase chain reaction.
 5. The method according toclaim 1, wherein said telomere length is determined from blood.
 6. Themethod according to claim 1, wherein said telomere length is determinedfrom lymphoid cells.
 7. The method according to claim 7, wherein saidlymphoid cells comprise T cells.
 8. The method according to claim 1,wherein said population is age matched with said individual organism. 9.The method according to claim 8, wherein said aged matched population iswithin about 10 human years of the age of said individual organism. 10.The method according to claim 9, wherein said aged matched population iswithin about 5 human years of the age of said individual organism. 11.The method according to claim 1 wherein said mortality is frominfectious diseases.
 12. The method according to claim 1, wherein saidmortality is from vascular disease.
 13. The method for predictingsurvival of an organism, said method comprising: a) determining the rateof telomere length decrease of said organism: and b) correlating saidrate of decrease with mortality risk associated with rate of telomerelength decrease in a population of the organism.